Project Summary/Abstract Cells that can count how many times they have cycled would be widely applicable both as research tools to study variations in cell cycle control and as control systems for activating synthetic genetic programs after a defined delay. We propose to develop a genetic device that achieves both programmable and accurate cell cycle counting in human cells. To achieve programmability so that the maximum count can be extended at will, we will use sequence programmable CRISPR guide RNAs to record and execute each counting step. To achieve accuracy, we will arrange these steps so that two CRISPR enzymes execute alternating steps, and we will separate these steps temporally by co-opting a natural circuit by which cells limit DNA replication to once per cell cycle. At the end of this study, we will have advanced genetic counter design to a stage where counters can be employed for certain basic science applications in human cells, and will have revealed the ways in which the counter can be further developed so that it is sufficiently robust for long-term biological and therapeutic applications.